US6599507B2 - Composition for the treatment of immune deficiencies and methods for its preparation and use - Google Patents

Composition for the treatment of immune deficiencies and methods for its preparation and use Download PDF

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US6599507B2
US6599507B2 US09/738,704 US73870400A US6599507B2 US 6599507 B2 US6599507 B2 US 6599507B2 US 73870400 A US73870400 A US 73870400A US 6599507 B2 US6599507 B2 US 6599507B2
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afp
complex according
aap
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Oleg Strelchenok
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Vivesto AB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to a new therapeutic composition, in particular an immunomodulating composition or a composition for uses in the treatment of immune deficiencies.
  • the invention also discloses methods for the preparation of said composition and methods for the therapeutic application of the same.
  • Alpha-fetoprotein a protein from mammalian fetal blood, has been held to be of a certain scientific interest since the moment it was first discovered in 1958.
  • the biological properties of this protein are subject of numerous investigations: there is however still no final answer as to the role of this protein in the organism.
  • unsaturated fatty acids such as arachidonic and docosahexaenoic acid and their metabolites are characterized as natural AFP ligands and can be detected as complexes with the given protein circulating in blood vessels.
  • AFP is not only a transport protein for unsaturated fatty acids. It has also been found that it forms complexes with bilirubin, retinoids and copper. In addition to the transport of low molecular weight substances AFP can take part in immune response regulation. Most of the studies on AFP's immunoregulating properties indicate that the protein has immunosuppressing features.
  • AFP obtained from fetal liver is characterized by a stronger suppression of mitogene-induced lymphocyte transformation in comparison with AFP, obtained from human blood of patients suffering from primary liver cancer.
  • AFP stimulates tissue regeneration after injuries.
  • AFP tended to decrease the inflammatory processes artificially simulated in animals supposedly by blocking the receptors of immunocompetent cells.
  • AFP is actively absorbed by growing and differentiating cells, and this process is controlled by the quantity of expressed AFP-receptors. It was found that intracellular AFP concentration increased simultaneously with the increase of the quantity of AFP-receptors on the surface of proliferating T-lymphocytes and malignant cells.
  • this protein functions as a shuttle-transporter, bringing the ligand inside the cell and then returning into intercellular liquid to repeat the cycle (Esteban C., et al., Int. J. Cancer, v.49, p. 425- 4 30, 1991).
  • the increase of the concentration of unsaturated fatty acids is of great importance as said acids are not only necessary structural components of the cell membrane, but also serve as an additional source of energy for the cells.
  • the metabolites of these acids in particular those of arachidonic acid, can act as secondary messengers, thus participating in the regulation of cellular growth and differentiation (Bevan S., et al., Nature (London), v. 328, p. 20, 1987).
  • Anandamide (arachidonyl-2-ethanolamid) is one of the recently discovered fatty acid metabolites. It is characterized by the high physiological effect targeted to brain. Anandamide is a novel lipid neurotransmitter first isolated from porcine brain. It has been shown to be a functional agonist for cannabinoid CB1 and CB2 receptors. Its presence results in many pharmacological effects caused by delta 9-tetra-hydrocannabinol (delta 9-THC). Anandamide parallels delta 9-THC in its specific interaction with the cannabinoid receptor and in the inhibition of adenylate cyclase. For many decades the mechanism of action of cannabinoid compounds, which are structurally similar to delta 9-THC, was unknown.
  • Cannabinoid derived drugs have been used for centuries for medicinal purposes. However, these drugs on the market today lack specificity and produce many side effects (Chakrabarti A., et al., Brain. Res. Bull., v.45, 30 p.67-74, 1998).
  • Anandamide can be formed enzymatically via two separate synthetic pathways in the brain: enzymatic condensation of the free arachidonic acid and ethanolamine; and formation of N-arachidonoyl phosphatidylethanolamine from phosphatidyletanolamine and arachidonic acid esterified at the 1-position of phosphatidylcholine, and subsequent release of anandamide from N-arachidonoyl phosphatydylethanolamine through the action of a phosphodiesterase (phospholipase D) (Suguira T., et al., Eur. J. Biochem., v.240. p.53-62, 1996).
  • phosphodiesterase phospholipase D
  • N-acyl-transferase catalyses the transfer of arachidonoyl residue onto the NH2 group of phosphatydylethanolamine. This enzyme is Ca2+dependent and is mostly localised in brain and testis. The pathway of anandamide formation is presented below:
  • R 1 and R 2 alkyl
  • N-arachidonoyl phosphatidylethanolamine could also be a substrate for phospholipase C (Brockerhoff H., Jensen R. G., Lipolytic enzymes, Academic press, New York-San Francisco-London, 1974). In this case the enzymatic reaction results in formation of N-arachidonoyl aminoethylphosphate (N-AAP).
  • N-AAP N-AAP
  • N-AAP can be protected by the protein molecule from the enzymatic influence in blood vessels as well as in the other biological liquids.
  • AFP depending on its origin and surrounding conditions, exerts different functions by different mechanisms. Firstly, there is a regulatory effect on the concentration of the unbound form of its various ligands (e.g. fatty acids, estrogens, phytosteroids). It is known, that fatty acids, in particular polyunsaturated fatty acids, modulate positively or negatively many steps of the action of various steroids and many enzymes involved in the transduction of membrane-triggered signals. Secondly, different conformations (holoforms) of AFP, depending on the nature and concentration of the ligand(s) bound to it, might influence the binding of the protein to specific receptor(s) and as a consequence influence it's/their biological properties (internalisation, action on the membrane signal transduction pathway). Thirdly, in addition to the mechanisms proposed above, the protein can exert effects associated with other signals, such as growth factors.
  • ligands e.g. fatty acids, estrogens, phytosteroids
  • the aim of the present invention is to make available a new pharmaceutical immunomodulating composition exhibiting improved properties, not only with respect to therapeutical properties such as efficacy and extent of application, but also pharmacological and technical properties such as ease of manufacture, storage, mixing and administration.
  • the present invention concerns a therapeutically useful complex, in particular an immunomodulating complex according to the attached claims.
  • the invention will be described in closer detail in the following description and examples.
  • the invention makes available an equilibrium reversible complex of alpha-fetoprotein and N-arachidonoyl aminoethylphosphate, in particular an equilibrated non-covalent complex of N-arachidonoyl aminoethylphosphate (N-AAP), a metabolite of arachidionic acid and alpha-fetoprotein (AFP) of high purity, for example AFP isolated from human cord blood with more than 99% purity.
  • N-AAP N-arachidonoyl aminoethylphosphate
  • AFP alpha-fetoprotein
  • the inventive complex may contain its components in highly varying molar ratios, such as from an equimolar ratio to a significant overabundance of N-AAP in relation to AFP. Normally, the complex contains from 1 up to 300 moles N-AAP per mole AFP.
  • the inventive complex may be obtained by adding an ethanol solution of N-AAP to a diluted water solution of AFP followed by ultrafiltration, said filtration resulting in concentrating the solution and removing the N-AAP that remained unbound to AFP.
  • the AFP concentration in solution varies from 0.1 up to 2 mg/ml and that of N-AAP—from 0.005 up to 30 mg/ml.
  • the protein is reversible linked not to one or several molecules of the ligand but surprisingly with a micelle, containing up to 300 molecules of N-AAP.
  • a micelle containing up to 300 molecules of N-AAP.
  • the natural AFP ligands like arachidonic, docosohexaenoic acids, etc. are sparingly soluble in water. If a concentrated ethanol solution of these substances is injected into water under special conditions one obtains a colloid solution.
  • the obtained colloid particles contain about from 50 to about 300 or more molecules of the lipid.
  • the addition of unsaturated fatty acids—AFP ligands to AFP water solution results in the formation of protein-lipid complexes.
  • the molecular weight of AFP incorporated in the complex with its natural ligand or its derivative increases by approx. 2 times, while gel-filtration of AFP with palmitoyl acid micelles did not result in the changes of elution volume in comparison with that for free AFP.
  • the micelle contained about 200-300 molecules of lipid.
  • the obtained preparations of AFP complexes with the micelles of its natural ligands are characterized as reversible protein-lipid complexes, but at the same time have the properties of proteoliposomes (Degrip W. J. Biochem J. Mar. 1. 330, p. 667-674, 1998).
  • the molecular weight of AFP incorporated in the complex with its natural ligand or its derivative increases by approx. 2-3 times.
  • the micelles contained 100-300 molecules of lipid.
  • the obtained preparations of AFP complexes with the micelles of its natural ligands or metabolites are characterised as reversible protein-lipid complexes, but at the same time have the properties of proteoliposomes.
  • AFP enter the cells via small vesicles and endosomes and move to multivesicular bodies and tubular vesicular elements located in the Golgi-centrosphere region to be finally recycled back into the medium (Geuskens M., et al. Microsc. Res. Tech. v.28, p. 297-307, 1994).
  • the reversible complexes of AFP with N-AAP penetrates into lymphocytes by means of AFP's receptor intermediated endocytosis.
  • the AFP/N-AAP complex inside lymphocytes could apparently regulate the synthesis of phospholipids as the structural components of cellular membrane.
  • N-AAP is a source of arachidonic acid which is further being incorporated into the phospholipid structures.
  • AFPIN-AAP complexes as well as their basic components on humoral immune response was estimated by counting the quantity of antibody-forming cells (AFC) in the spleen. It has been experimentally proved that N-AAP in itself does not exhibit immunogenic activity. The relative amount of AFC cells on the 5-th day after N-AAP injection to the animals immunized with sheep erythrocytes was not significantly changed in comparison with control series. Administration of the same dosage of the inventive AFP/N-AAP complex (AFPIN-AAP ratio 1:200) resulted in that the relative amount of AFC increased 87% and the total AFC amount increased 162% on the 5-th day after injection in comparison with the amount of cells in animals immunized only with sheep erythrocytes.
  • AFP alone administered in corresponding doses reduces or does not significantly effect the immunogenic characteristics of sheep erythrocytes in mice.
  • AFP was isolated from human cord blood by immunoaffinity chromatography on monoclonal antibodies against AFP immobilised on Sepharose®, immunoaffinity chromatography on polyclonal antibodies to the proteins of normal human blood and gel-filtration on Sephacryl® S-200.
  • the AFP preparation thus obtained was more than 99% purity and did not contain low molecular weight impurities and retained completely its biological activity.
  • AFP may be purified and/or modified AFP from other mammals, for example from genetically modified mammals, or from cell cultures.
  • the AFP is biotechnologically manufactured using a cell culture of genetically modified cells expressing human AFP. With knowledge of the nucleotide sequence coding for human AFP, this can be inserted in a host, together with necessary promoters and other sequence information, for example sequences influencing the extracellular expression of AFP.
  • the AFP is collected from the cell culture and purified by chromatography, and may be further purified by gelfiltration. In any case, the production method must involve steps, which guarantee that the final product is free from pyrogens and possible viral or bacterial contaminants. Suitable production methods can for example be found in the field of interferon production.
  • the AFP/N-AAP complex is used as a therapeutic agent either as such, or used for the manufacture of a therapeutical preparation, possibly containing other agents.
  • the inventive complex is particularly suitable as an immunostimulating agent, e.g. for the treatment of immune deficiencies.
  • the complex can also be used for the manufacture of an immunostimulating preparation.
  • the inventive complex is used for the treatment of immune disorders associated with cancer therapy.
  • the inventive complex can also be used for the manufacture of a pharmaceutical preparation for the treatment of cancer. Examples of such disorders or immune deficiencies occurring as a consequence of cancer treatment, include neutropenia.
  • the inventive complex can also be used as a prophylactic agent in patients susceptible for infections, or for the manufacture of a pharmaceutical composition for the treatment of such patients.
  • the invention also concerns methods for the treatment of immune deficiencies, wherein an equilibrium reversible complex according to the present invention is administered to a mammal.
  • said complex is administered intravenously.
  • the active composition may be administered intravenously.
  • the composition can be prepared as a sterile powder for extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent allowing for easy handling by syringe and similar devices. Further, the preparation must be stable under conditions of manufacture and storage and must be protected against the contaminating action of micro-organisms such as bacteria and fungi.
  • Sterile injectable solutions are prepared by incorporating AFP and N-AAP in a required amount of water, ultrafiltration (concentration) of the solution followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying or suitable freeze-drying techniques that yield a powder of the active ingredients from previously sterile filtered solutions thereof.
  • a sterilized preparation is first injected in a physiological saline solution (100-500 ml) or in a protein (albumin) solution then intravenously administered to a patient.
  • a lyophilized preparation it should be previously dissolved in 2-10 ml of sterilized solution (distilled water, physiological saline solution or albumin solution) whereupon the obtained sterilized solution is added to 100-500 ml physiological solution or protein solution for intravenous introduction, same as in case of using the solutions.
  • the therapeutic effective doses are in interval from 2 mg/kg to 7 mg/kg for N-AAP and from 0.2 mg/kg to 0.7 mg/kg for AFP.
  • the immunoaffinity matrix for AFP isolation was prepared by means of immobilising anti-AFP monoclonal antibodies isolated from mouse ascite liquid (AFP-Ab) on BrCN-Sepharose®. 30 mg AFP-Ab dissolved in 10 ml bicarbonate buffer were then added to 25 ml condensed BrCN-Sepharose® and incubated for 1 hr at room temperature. The obtained AFP-Ab-Sepharose® was washed with 0.5 l bicarbonate buffer. The affinity matrix contained about 1 mg AFP-Ab per 1 ml condensed gel.
  • the immunoaffinity matrix for AFP purification of serum proteins was prepared by means of immobilizing polyclonal antibodies against normal human serum proteins on BrCN-Sepharose®. 30 mg antibodies dissolved in 10 ml bicarbonate buffer were then added to 25 ml condensed BrCN-Sepharose® and incubated for 1 hr at room temperature. The obtained immunosorbent was washed with 0.5 l bicarbonate buffer. The final affinity matrix contained about 1 mg immobilized antibodies per 1 ml condensed gel.
  • N-AAP affinity to human AFP a competitive substitution of [5, 6, 8, 9, 11, 12, 14, 15- 3 H] arachidonic from the protein's binding site was used.
  • tubes containing 0.05 nM AFP in 1 ml 0.1 M bicarbonate buffer and 0.7 nM [3H] arachidonic acid and the increasing amounts of arachidonic acid or N-AAP (5-5000 nM) were added.
  • the tubes were incubated for 2 hr at room temperature.
  • To separate protein-bound and free fractions of [3H] arachidonic acid 0.5 ml of 0.5% suspension of activated carbon was added to each tube and incubated at 4° C. for 30 min. The carbon was sedimented by centrifugation at 3000 g, aliquots were added to 10 ml scintillating mixture and the vials were measured in beta-counter.
  • the binding parameters of arachidonic acid and N-AAP and the number of binding sites per protein molecule were calculated according to Scatchard (Scatchard O., Ann. N.Y. Acad. Sci. 51., p 660-664, 1949).
  • AFP 50 mg AFP (0.75 mmol) was dissolved in 150 ml physiological solution. 35 mg ( ⁇ 75 mmol) N-AAP dissolved in 5 ml ethanol was added to the obtained solution. The mixture was incubated for 30 min at room temperature (20-25° C.). The obtained complex of N-AAP with AFP was concentrated to 10 ml using Sartocon® Micro “Sartorius” for high-molecular weight compounds with a 20.000 Da membrane cut off. The final solution was sterilized with a syringe tip Minisart® SRP Syringe Filter, “Sartorius” a membrane of 0.22 ⁇ pore size. A sterilized concentrated preparation was distributed in 10 vials of 1 ml each. The vials were blown with argon stream, closed firmly and stored at 4-8° C.
  • AFP was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in a dose of 0.009 mg per capita. Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita) to both control and experimental animals. The control animals were also injected with the equal volume of NaCl isotonic solution intravenously.
  • AFP The effect of AFP on humoral immune response was analyzed by counting the quantity of AFC in the spleen according to Cunningham (Cunningham A. J., Nature, v.207, p. 1106-1107, 1965) (per 10 6 spleen cells and per spleen).
  • N-AAP was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in a dose of 0.09 mg per capita Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita). Control series were injected with 0.15 ml NaCl isotonic solution intravenously.
  • the effect of N-AAP on humoral immune response was analysed by counting the quantity of AFC in the spleen according to Cunningham (per 10 6 spleen cells and per spleen).
  • the relative amount of AFC on the 5-th day after injection was 366.2 ⁇ 40.8 for control animals and 342.2 ⁇ 28.5 for experimental series, P>0.05.
  • the total AFC amount was 27.2 ⁇ 5.7 10 3 for control group and 36.0 ⁇ 8.7 10 3 for experimental series, P>0.05.
  • the obtained data shows that N-AAP itself has no immunogenic activity in itself.
  • AFP/N-AAP complex 0.15 ml AFP/N-AAP complex was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in a dose of 0.009 mg AFP and 0.09 mg N-AAP per capita. Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita). Control series were intravenously injected with equal volumes of NaCl isotonic solution. The effect of the AFP/N-AAP complex on humoral immune response was analyzed by counting the quantity of AFC in the spleen according to Cunningham (per 10 6 spleen cells and per spleen).
  • the relative amount of AFC on the 5-th day after injection was 366.2 ⁇ 40.8 for control animals and 686.7 ⁇ 89.5 for experimental series, P ⁇ 0.05.
  • the total AFC amount was 27.2 ⁇ 5.3 10 3 for control group and 71.0 ⁇ 18.7 10 3 for experimental series, P ⁇ 0.05.
  • the relative amount of AFC on the 5-th day after injection increased 87%, and the total AFC amount increased 162% in comparison with the amount of cells in the animals immunized only with sheep erythrocytes.
  • the data obtained proved that in those animals receiving AFP (0.009 mg per capita, see example 5) combined with sheep erythrocytes injections the relative amount of AFC increased 169% and total AFC amount 203%.
  • AFP/N-AAP complex 0.15 ml AFP/N-AAP complex was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in a dose of 0.009 mg AFP and 0.045 mg N-AAP per capita. Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita). Control series were intravenously injected with equal volumes of NaCl isotonic solution. The effect of the AFP/N-AAP complex on humoral immune response was analyzed by counting the quantity of AFC in the spleen according to Cunningham (per 10 6 spleen cells and per spleen).
  • the relative amount of AFC on the 5-th day after injection was 366.2 ⁇ 40.8 for control animals and 476.2 ⁇ 28.1 for experimental series, P ⁇ 0.05.
  • the total AFC amount was 27.2 ⁇ 5.7 10 3 for control group and 48.0 ⁇ 6.2 10 3 for experimental series, P ⁇ 0.05.
  • the apparent amount of AFC on the 5-th day after injection increased 30%, and total AFC amount increased 79% in comparison with amount of cells in the animals immunized only with sheep erythrocytes.
  • the data obtained proved that in those animals receiving AFP (0.009 mg per capita, see example 5) combined with sheep erythrocytes injections the apparent amount of AFC increased 86.6% and total AFC amount 103.4%.
  • AFP/N-AAP complex 0.15 ml AFP/N-AAP complex was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in a dose of 0.009 mg AFP and 0.135 mg N-AAP per capita. Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita) Control series were intravenously injected with equal volumes of NaCl isotonic solution. The effect of the AFP/N-AAP complex on humoral immune response was analyzed by counting the quantity of AFC in the spleen according to Cunningham (per 10 6 spleen cells and per spleen).
  • the relative amount of AFC on the 5-th day after injection was 366.2 ⁇ 40.8 for control animals and 543.2 ⁇ 50.2 for experimental series, P ⁇ 0.05.
  • the total AFC amount was 27.2 ⁇ 5.7 10 3 for the control group and 55.3 ⁇ 5.3 ⁇ 10 3 for the experimental series, P ⁇ 0.05.
  • the relative amount of AFC on the 5-th day after injection increased 48.3%, and total AFC amount increased 103% in comparison with amount of cells in the animals immunized only with sheep erythrocytes.
  • the data obtained proved that in those animals receiving AFP (0.009 mg per capita, see example 5) combined with sheep erythrocyte injections the relative amount of AFC increased 112.8% and total AFC amount 134.3%.
  • the desired phosphorylated N-acylaminoalcohol was eluted from the column with chloroform—methanol (70-60:30-40, v/v).
  • the composition of the eluates was controlled by TLC on Silica gel 60 plates [chloroform-methanol-NH 3 aq (9:7:2,v/v/v)] using a molybdate spray for detecting the spots.
  • the appropriate fractions were combined, evaporated to dryness in vacuo and residue was dissolved in 1 ml tetrahydrofuran. That solution was added, dropwise over a period of 5 min, to a cooled (ice-bath), stirred 1.5 M NaOH aq (4 ml).
  • Sephacryl® S-300-HR and gel filtration molecular weight markers i.e. proteins having a range of molecular weights from 29 kD to 700 kD (the MW-GF-1000 kit) were purchased from Sigma Chemical Co. [5, 6, 8, 9, 11, 12, 1 15- 3 H] arachidonic acid was purchased from Amersham International, UK.
  • Rat AFP was isolated from neonatal rat serum. Monospecific anti-rat serum alpha-fetoprotein IgG was coupled to cyanogen bromide-activated Sepharose 4B (4.5 mg/ml packed volume of gel) to yield an immunoaffinity matrix. Acidic elution condition were as described previously (Calvo, M., et al., J. Chromatogr. V.328, p.392-395, 1985). Similar to human AFP the complexes of rat AFPIN-AAP reveal strong effect on humoral immune response in mice (example 11).
  • AFP/N-AAP complex was administered intravenously to 10 mouse males of CBA line (weight 18-22 g) in the dose of 0.009 mg rat AFP and 0.09 mg N-AAP per capita. Simultaneously 5% sheep erythrocytes suspension was injected peritoneally (0.2 ml per capita). Control series were injected intravenously with equal volumes of isotonic NaCl solution. The effect of rat AFP/N-AAP complex on humoral immune response was analyzed by counting the quantity of AFC in the spleen according to Cunningham (per 10 6 spleen cells and per spleen).
  • the relative amount of AFC on the 5-th day after injection was 338.8 ⁇ 67.9 for control animals and 659 ⁇ 38.4 for experimental series, P ⁇ 0.05.
  • the total AFC amount was 51.2 ⁇ 13.5 ⁇ 10 3 for the control group and 89.4 ⁇ 11.8 ⁇ 10 3 for the experimental series, P ⁇ 0.05.
  • Rat AFP exhibits no immunogenic activity.
  • the relative amount of AFC was 425.1 ⁇ 42.1 for control animals and 428.1 ⁇ 11.5 for experimental series, P>0.05.
  • the total AFC amount was 65.4 ⁇ 6.8 ⁇ 10 3 for the control group and 60.3 ⁇ 13.5 ⁇ 10 3 for the experimental series, P>0.05.
  • the relative amount of AFC on the 5-th day after injection increased 195%, and total AFC amount increased 175% in comparison with amount of cells in the animals immunized only with sheep erythrocytes.
  • N-AAP The toxicity of N-AAP was studied in random breed male mice (19-24 g). A water solution of N-AAP was injected once slowly in the tail vein of mice in a range of doses 40-70 mg/kg. Each group of mice included 6 animals, at least The assessment of acute toxicity was carried out according to the method of Litchfield et al. (Litchfield J. T., Wilcoxon F., J. Pharmacol. Exptl. Therap., Vol.96, P. 99-103, 1949). The acute toxicity of N-AAP was shown to be as follows:
  • the therapeutic index is 10.
  • the present inventor tested AFP/N-AAP complexes at the ratios—1/100, 1/200 and 1/300 in order to estimate the influence of these complexes on humoral immune response in mice (examples 8-10). All presented complexes reveal biological activity in all experiments. The complex AFP/N-AAP was shown to have a more powerful effect on humoral immune response, at the ratio 1:200 mole/mole.

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ATE243701T1 (de) * 1999-03-16 2003-07-15 Ardenia Investments Ltd Polyungesättigte fettsäurederivate und deren verwendung in der krebsbehandlung
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US8071547B2 (en) * 2005-11-15 2011-12-06 Constab Pharmaceutical, Inc. Compositions of alpha-fetoprotein and inducers of apoptosis for the treatment of cancer

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SE9802162D0 (sv) 1998-06-17
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ATE314094T1 (de) 2006-01-15
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